Optical transfection

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Optical transfection is a biomedical technique that entails introducing nucleic acids (i.e. genetic material such as DNA) into cells using light. All cells are surrounded by a plasma membrane, which prevents many substances from entering or exiting the cell. Lasers can be used to burn a tiny hole in this membrane, allowing substances to enter. This is tremendously useful to biologists who are studying disease, as a common experimental requirement is to put things (such as DNA) into cells.

Contents

Typically, a laser is focussed to a diffraction limited spot (~1 μm diameter) using a high numerical aperture microscope objective. The plasma membrane of a cell is then exposed to this highly focussed light for a small amount of time (typically tens of milliseconds to seconds), generating a transient pore on the membrane. The generation of a photopore[ check spelling ] allows exogenous plasmid DNA, RNA, organic fluorophores, or larger objects such as semiconductor quantum nanodots to enter the cell. In this technique, one cell at a time is treated, making it particularly useful for single cell analysis.

This technique was first demonstrated in 1984 by Tsukakoshi et al., who used a frequency tripled Nd:YAG to generate stable and transient transfection of normal rat kidney cells. [1] Since this time, the optical transfection of a host of mammalian cell types has been demonstrated using a variety of laser sources, including the 405 nm continuous wave (cw), [2] 488 nm cw, [3] or pulsed sources such as the 800 nm femtosecond pulsed Ti:Sapphire [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] or 1064 nm nanosecond pulsed Nd:YAG. [14] [15]

Terminology

The meaning of the term transfection has evolved. [16] The original meaning of transfection was "infection by transformation", i.e. introduction of DNA (or RNA) from a prokaryote-infecting virus or bacteriophage into cells, resulting in an infection. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA (or other nucleic acid species such as RNA or SiRNA).

Because of this strict definition of transfection, optical transfection also refers only to the introduction of nucleic acid species. The introduction of other impermeable compounds into a cell, such as organic fluorophores or semiconductor quantum nanodots is not strictly speaking "transfection," and is therefore referred to as "optical injection" or one of the many other terms now outlined.

The lack of a unified name for this technology makes reviewing the literature on the subject very difficult. [17] Optical injection has been described using over a dozen different names or phrases (see bulleted lists below). Some trends in the literature are clear. The first term of the technique is invariably a derivation of word laser, optical, or photo, and the second term is usually in reference to injection, transfection, poration, perforation or puncture. Like many cellular perturbations, when a single cell or group of cells is treated with a laser, three things can happen: the cell dies (overdose), the cell membrane is permeabilised, substances enter, and the cell recovers (therapeutic dose), or nothing happens (underdose). There have been suggestions in the literature to reserve the term optoinjection for when a therapeutic dose is delivered upon a single cell, [18] [19] [20] and the term optoporation for when a laser generated shockwave treats a cluster of many (10s to 100s) cells. [18] [19] [14] [20] The first definition of optoinjection is uncontroversial. The definition of optoporation, however, has failed to be adopted, with a similar number of references using the term to denote the dosing of single cells [3] [5] [15] [21] as those using the term to denote the simultaneous dosing of clusters of many cells [18] [19] [14] [20]

As the field stands, it is the opinion of the authors of a review article on the subject [17] that the term optoinjection always be included as a keyword in future publications, regardless of their own naming preferences.

Terms agreed by consensus

Terms under deliberation

Some of the above was reproduced with permission from. [17]

Methods

A typical optical transfection protocol is as follows: [11] 1) Build an optical tweezers system with a high NA objective 2) Culture cells to 50-60% confluency 3) Expose cells to at least 10 μg/mL of plasmid DNA 4) Dose the plasma membrane of each cell with 10-40 ms of focussed laser, at a power of <100 mW at focus 5) Observe transient transfection 24-96h later 6) Add selective medium if the generation of stable colonies is desired

See also

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References

  1. ↑ Tsukakoshi, M.; Kurata, S.; Nomiya, Y.; Ikawa, Y.; Kasuya, T. (1984). "A novel method of DNA transfection by laser microbeam cell surgery". Applied Physics B: Photophysics and Laser Chemistry. 35 (3). Springer Science and Business Media LLC: 135–140. Bibcode:1984ApPhB..35..135T. doi:10.1007/bf00697702. ISSN   0721-7269. S2CID   123250337.
  2. ↑ Paterson, L.; Agate, B.; Comrie, M.; Ferguson, R.; Lake, T. K.; et al. (2005). "Photoporation and cell transfection using a violet diode laser". Optics Express. 13 (2). The Optical Society: 595–600. Bibcode:2005OExpr..13..595P. doi: 10.1364/opex.13.000595 . ISSN   1094-4087. PMID   19488389.
  3. 1 2 3 Palumbo, Giuseppe; Caruso, Matilde; Crescenzi, Elvira; Tecce, Mario F.; Roberti, Giuseppe; Colasanti, Alberto (1996). "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation". Journal of Photochemistry and Photobiology B: Biology. 36 (1). Elsevier BV: 41–46. doi:10.1016/s1011-1344(96)07335-6. ISSN   1011-1344. PMID   8988610.
  4. ↑ Tsampoula, X.; Taguchi, K.; Čižmár, T.; Garces-Chavez, V; Ma, N.; et al. (2008-10-10). "Fibre based cellular transfection". Optics Express. 16 (21). The Optical Society: 17007–13. Bibcode:2008OExpr..1617007T. doi: 10.1364/oe.16.017007 . ISSN   1094-4087. PMID   18852810.
  5. 1 2 3 Uchugonova, Aisada; König, Karsten; Bueckle, Rainer; Isemann, Andreas; Tempea, Gabriel (2008-06-11). "Targeted transfection of stem cells with sub-20 femtosecond laser pulses". Optics Express. 16 (13). The Optical Society: 9357–64. Bibcode:2008OExpr..16.9357U. doi: 10.1364/oe.16.009357 . ISSN   1094-4087. PMID   18575499.
  6. ↑ Brown, C. T. A.; Stevenson, D. J.; Tsampoula, X.; McDougall, C.; Lagatsky, A. A.; et al. (2008). "Enhanced operation of femtosecond lasers and applications in cell transfection". Journal of Biophotonics. 1 (3). Wiley: 183–199. doi: 10.1002/jbio.200810011 . ISSN   1864-063X. PMID   19412968. S2CID   10801756.
  7. ↑ Baumgart, J.; Bintig, W.; Ngezahayo, A.; Willenbrock, S.; Murua Escobar, H.; Ertmer, W.; Lubatschowski, H.; Heisterkamp, A. (2008). "Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells". Optics Express. 16 (5). The Optical Society: 3021–31. Bibcode:2008OExpr..16.3021B. doi: 10.1364/oe.16.003021 . ISSN   1094-4087. PMID   18542388.
  8. ↑ Lei, Ming; Xu, Hanpeng; Yang, Hao; Yao, Baoli (2008). "Femtosecond laser-assisted microinjection into living neurons". Journal of Neuroscience Methods. 174 (2). Elsevier BV: 215–218. doi:10.1016/j.jneumeth.2008.07.006. ISSN   0165-0270. PMID   18687359. S2CID   10242427.
  9. ↑ Tsampoula, X.; Garcés-Chávez, V.; Comrie, M.; Stevenson, D. J.; Agate, B.; Brown, C. T. A.; Gunn-Moore, F.; Dholakia, K. (2007-07-30). "Femtosecond cellular transfection using a nondiffracting light beam". Applied Physics Letters. 91 (5). AIP Publishing: 053902–053903. Bibcode:2007ApPhL..91e3902T. doi:10.1063/1.2766835. ISSN   0003-6951.
  10. ↑ Peng, Cheng; Palazzo, Robert E.; Wilke, Ingrid (2007-04-03). "Laser intensity dependence of femtosecond near-infrared optoinjection". Physical Review E. 75 (4). American Physical Society (APS): 041903. Bibcode:2007PhRvE..75d1903P. doi:10.1103/physreve.75.041903. ISSN   1539-3755. PMID   17500917.
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  12. ↑ Barrett, Lindy E; Sul, Jai-Yoon; Takano, Hajime; Van Bockstaele, Elisabeth J; Haydon, Philip G; Eberwine, James H (2006-05-23). "Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor". Nature Methods. 3 (6). Springer Science and Business Media LLC: 455–460. doi:10.1038/nmeth885. ISSN   1548-7091. PMID   16721379. S2CID   10536176.
  13. ↑ Tirlapur, Uday K.; König, Karsten (2002). "Targeted transfection by femtosecond laser". Nature. 418 (6895). Springer Science and Business Media LLC: 290–291. doi:10.1038/418290a. ISSN   0028-0836. PMID   12124612. S2CID   4370674.
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  16. ↑ "Transfection" at Dorland's Medical Dictionary
  17. 1 2 3 Stevenson, David J.; Gunn-Moore, Frank J.; Campbell, Paul; Dholakia, Kishan (2010-01-11). "Single cell optical transfection". Journal of the Royal Society Interface. 7 (47). The Royal Society: 863–871. doi:10.1098/rsif.2009.0463. ISSN   1742-5689. PMC   2871806 . PMID   20064901.
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